In addition to the extracellular proteins secreted by known secretory pathways, a number of cytoplasmic proteins without predicable or known signal sequences or secretory motifs have been found in the extracellular milieu, and were consequently classified as non-classically secreted proteins. Non-classical protein secretion is considered to be a general, conserved cellular phenomenon in both eukaryotes and prokaryotes. There are several research hotspots on the non-classical protein secretory pathway, and the most important two of them are the recognition principle of substrate proteins and possible secretory mechanisms. To date, researchers have made some progress in understanding the characteristics of these proteins. For example, it was discovered that many non-classically secreted proteins exist and are secreted in multimeric form. Some of these proteins prefer to be clustered and exported at the poles and the septum of the cell. The majority of these proteins play different functions when they are in the intra- and extracellular environments, and several of their functions are related to survival and pathogenicity. Furthermore, non-classically secreted proteins can be used as leading proteins to guide a POI (protein of interest) out of the cells, which provides a novel strategy for protein secretion with potential applications in the industry. Summarizing these findings, this review emphasizes the hot spots related to non-classically secreted proteins in bacteria, lists the most important hypotheses on the selection and secretion mechanisms of non-classically secreted proteins, and put forward their potential applications.